Vanadyl organo-mineral compound, method for obtaining such a compound, pharmaceutical composition containing this organo-mineral compound

ABSTRACT

The present invention concerns a vanadyl organo-mineral compound which is in the form of a complex of vanadyl and of cystein, having the following general formula: ##STR1## wherein, either x is 1 and y is 0, or x is 0 and y is 1, and wherein n, p and m are respectively integers with a value of 1 or 2.

This is a division of application Ser. No. 07/230,520 filed Aug. 10,1988, U.S. Pat. No. 5,023,358.

The present invention concerns generally a vanadyl organo-mineralcompound. It has essentially as an object a complex comprising vanadyl,methods for obtaining such a complex and a pharmaceutical compositioncontaining the complex.

The invention finds application in particular in the pharmaceuticalindustry, for the manufacture of anti-diabetic medicines which can betaken orally.

Many compounds are known for their anti-diabetic activity for patientssuffering from insulin-depending diabetes. However, the conventionalcompositions on the basis of these compounds are taken parenterally.

A few compounds have been discovered which are taken orally. Forexample, compounds on the basis of vanadate which is the oxydized formof vanadium, such as sodium vanadate which, when taken orally, has ananti-diabetic effect, are known. Indeed, in "Clayton, Arun, and McNeill, Science, Mar. 22, 1985, vol. 227, pages 1474-1477", Mc Neill etal. have shown that, by adding sodium vanadate to the drink water ofdiabetic rats, the glycemia of the rats is normalized and the impairingof the cardiac function associated with diabetes is prevented.

However, the sodium vanadate presents a digestive toxicity due to itsvery weak absorption which is of about 1 to 3%. The vanadate (V⁵⁺)presents a number of properties, some of which cause this non-digestivetoxicity, for example the property of inhibiting the Na⁺ /K⁺ ATP aseenzyme in the absorption mechanisms.

Besides, it is known that on the intra-cellular scale, the vanadiumreveals its anti-diabetic activity under the vanadyl (V⁴⁺) form byfacilitating or inducing the phosphorylation of the insulin receptor

Now, the vanadyl sulfate VOSO₄ administered orally to diabetic rats hasan anti-diabetic effect but presents an acute toxicity.

The present invention has as an object to provide a new vanadylorgano-mineral compound which does not present the above mentioneddrawbacks in the case of the use of the sodium vanadate and of thevanadyl sulfate, and which is both non-toxic and active as ananti-diabetic compound. Such a compound, which remains in the V⁴⁺ formin the blood, is better absorbed during the digestion and can diffuse inthe tissues in an active form.

According to the present invention, this object is attained by a vanadylorgano-mineral compound in the form of a complex of vanadyl and ofcystein. This compound has the following general formula: ##STR2##wherein, either x is 1 and y is 0, or x is 0 and y is 1, and wherein n,p and m are integers respectively equal to 1 or 2.

Such a compound, due to the complexing of the vanadyl ion by a cysteinderivative, shows a higher diabetic activity and a lower toxicity thanthe orally administered anti-diabetic compounds known in the state ofthe art.

According to a feature of the invention, in the aforesaid formula, x is1, y is 0, p is 1 and Y is hydrogen and Z is a minus charge. Such acomplex has a free amine group.

According to an advantageous feature of the invention, in the aforesaidformula n is 1 and m is 2. The complex is then in the form of abimolecular ligand.

According to still another advantageous feature of the invention, in theaforesaid formula n is 2 and m is 1. The complex is then in the form ofa monomolecular ligand.

According to another feature of the invention, in the aforesaid formulax is 0, y is 1, n is 1, X is a O⁻ group and Z is hydrogen. This secondtype of complex has a free carboxyl group.

According to an advantageous feature of the invention, in the aforesaidformula p is 1 and m is 2. The complex is then in the form of abimolecular ligand.

According to still another advantageous feature of the invention, in theaforesaid formula p is 2 and m is 1. The compound is then amonomolecular ligand.

According to another feature of the invention, in the aforesaid formulax is 0, y is 1, Y is a CH₂ group and Z is a minus charge.

According to a characteristic of the invention, in the aforesaid formulan is 1 and p is 1 and m is 2. This compound is a monomolecular ligandwith substituted amine and carboxyl groups.

An other object of the invention consists of a method for preparing thevanadyl organo-mineral compound which is in the form of a vanadyl andcystein complex of the formula (I) and which comprises one free aminegroup or substituted amine and carboxyl groups.

Such a method is characterized:

by reacting a mono- or bifunctional amine or a mono- or bifunctionalalcohol, or an amine-alcohol, with the cystein or a derivative thereof,which is protected the amine function and on the thiol function by atertiobutyloxycarbonyl group in the presence ofdicyclohexylcarbodiimide/hydroxybenzotriazole,

by eliminating the said tertiobutyloxycarbonyl group by acidolysis,

by adding a vanadylsulfate dissolved in water, under a nitrogenatmosphere, to the solution of the hydrochloride of the cysteinderivative in a dimethylformamide-borate buffer mixture at a pH of about10, with a cystein:vanadyl ratio of about 5:1,

by recovering the precipitated complex, and

by washing with water and drying the said precipitate.

A second method for preparing the compound having one free amine groupor substituted amine and carboxyl groups is characterized:

by reacting the cystein or a derivative thereof with vanadyl sulfate ata pH of about 7 in water,

by recovering the complex obtained after evaporation,

by redissolving the said complex in dimethylformamide,

by coupling with a mono- or bifunctional amine, a mono- or bifunctionalalcohol or an amine-alcohol, through the medium ofdimethylaminopropylethylcarbodiimide, and

by recovering the complex after vacuum evaporation of the solvent, andby washing the said complex with ether and with water .

The invention has also as an object a pharmaceutical composition, inparticular an anti-diabetic composition, comprising as an activesubstance the compound such as defined previously.

Such a composition has an anti-diabetic activity and is non-toxic, itcan be taken orally, which presents a considerable advantage for thepatients suffering from insulin-depending diabetes.

The invention will be better understood and other objects, features anddetails thereof will appear more clearly as the following explanatorydescription proceeds, with reference to the illustrative and nonlimitative examples.

The new vanadyl organo-mineral compound results from the complexing of avanadyl and of a cystein or cystein derivative and corresponds to thegeneral formula (I).

More precisely, this laevo-rotary (L) or dextro-rotary (D) complex has afree amine group or a free carboxyl group or substituted amine andcarboxyl groups.

a) In the case where the ligand comprises a free amine group, it can bebimolecular on the one hand and has the following general formula:##STR3##

It may be monomolecular, on the other hand, and has therefore thefollowing formula: ##STR4##

b) In the case where the ligand comprises a free carboxyl group, it canbe, similarly, bimolecular on the one hand, and has as a generalformula: ##STR5##

On the other hand, the ligand can be monomolecular and has as a generalformula: ##STR6##

c) in the case where the ligand comprises substituted amine and carboxylgroups, it is in the monomolecular form and has the following generalformula: ##STR7##

In formulae (II) and (VI), X is a --OR group or a --NHR (amine) group,in which the R moiety is an aryl or aralkyl group or an alkyl groupother than methyl.

Therefore, in the case where X is a --NHR group, the organo-mineralcompound is for example a complex of vanadyl with an amide of cysteinsuch as in particular a butylamide of cystein or an octylamide ofcystein. The vanadyl-octylamide (L) complex of the cystein isillustrated by the following formula: ##STR8##

Moreover, in the case where X is a --OR group, the organo-mineralcompound is for example a complex of vanadyl with an ester of thecystein such as in particular an octylester of cystein or a butylesterof cystein.

Besides, in the formula (III), X corresponds to one of the followinggroups: ##STR9## wherein R is an aryl or aralkyl group or an alkyl groupother than butyl.

In formula (IV), Y corresponds to a R-CO-(ketone) group in which the Rmoiety is an alkyl, aryl or aralkyl group.

In formula (V), Y corresponds to one of the following groups: ##STR10##wherein R is also an alkyl, aryl or aralkyl group.

By way of example, the preparation of the compounds, namely the vanadylcomplexes of the formulae (II), (III), (IV), (V) and (VI) will be giventhereafter.

(i) A method for preparing the compound (II) or (VI) consists in:

reacting a monofunctional amine or a monofunctional alcohol with thecystein or a derivative thereof protected on the amine function and onthe thiol function by a BOC or tertiobutyloxycarbonyl group in thepresence of dicyclohexylcarbodiimide/hydroxybenzotriazole,

eliminating the said BOC or tertiobutyloxycarbonyl group by acidolysiswith the aid of hydrochloric acid and dioxan,

adding vanadylsulfate which is dissolved in water, under a nitrogenatmosphere, to the solution of the hydrochloride of the cysteinderivative in a dimethylformamide-borate buffer mixture at a pH of about10 with a cystein/vanadyl ratio of 5/1,

then, stirring the mixture for 2 hours in a nitrogen atmosphere,

recovering the complex formed by precipitation, by means of afiltration, and

washing with water and drying the precipitate.

The product obtained presents the following characteristics. It isinsoluble in water and in ethylether. On the other hand, it is solublein DMF or dimethylformamide and in methanol.

The example 1 below illustrates an embodiment of this method in the caseof the preparation of a complex of vanadyl and of an octylamide of thecystein.

(ii) The preparation of the compound of formula (III) consists inreacting a bifunctional amine or a bifunctional alcohol or anamine-alcohol with the cystein or a derivative thereof. The remainingprocedure is identical to that of the above described method ofpreparation.

(iii) Another method for preparing the compound of formula (II) or (VI),consists in:

reacting the cystein or derivative thereof with vanadyl sulfate at a pHof about 7 in water,

recovering the complex obtained after evaporation,

redissolving the complex formed in dimethylformamide as above,

coupling with a monofunctional amine or with a monofunctional alcoholthrough the medium of dimethylaminopropylethylcarbodiimide, and

recovering the complex after vacuum evaporation of the solvent, washingwith ether and with water, dissolution in methanol, evaporation andreprecipitation by ethylether.

This method is illustrated by example 2 below in the case of thepreparation of a complex of vanadyl with cystein octylamide.

(iv) The preparation of the compound (III) proceeds with the same stepsas the method mentioned above for the preparation of compounds (II) and(VI), however, as starting product, a bifunctional amine or abifunctional alcohol or an amine-alcohol is reacted with the cystein ora derivative thereof.

(v) The method of preparing the compound of formula (IV) where Ycorresponds to the R-CO-group, consists in:

coupling the activated derivative (ester or chloride) of the R-COOH acidwith the cystein previously protected on its thiol function by a BOCgroup,

deprotecting then the thiol function by acidolysis; and

complexing the obtained N-acylated derivative with the vanadyl in aDMF-water medium at a pH of 10, such a medium containing the vanadylsulfate.

(vi) Finally, the method for preparing the compound of formula (V) whereY corresponds to: ##STR11## consists in:

coupling the activated derivative (ester or chloride) of the diacid##STR12## with the cystein previously protected on the thiol function bya BOC group,

deprotecting the thiol function by acidolysis,

reducing the N-acylated derivative thus obtained; and

then complexing this N-acylated derivative in a DMF-water medium at a pHof 10, in the presence of vanadyl sulfate.

EXAMPLE 1

According to the above method (i), vanadyl sulfate dissolved in water isreacted with a solution of the hydrochloride of the cystein octylamidehaving the following formula: ##STR13## in an atmosphere of nitrogen ina dimethylformamide-borate buffer mixture (pH of about 10) with acystein to vanadyl molar ratio of 5:1. The product is obtained byprecipitation.

By means of a quantitative analysis of vanadium by atomic absorption,there is obtained a stoichiometry of 2:1 of the cystein with respect tovanadyl and a weight percentage of vanadium of about 9.6%+0.2%.

The product thus obtained is a bimolecular complex or ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically shows by analysis that vanadium is in the vanadylform and

FIG. 2 shows the effect, graphically, of administration of compound(II).

Moreover, the analysis by electronic paramagnetic resonance withreference to FIG. 1, presents resonance bands with a hyperfinestructure. It results from this analysis that vanadium is in the VO²⁺vanadyl form.

Consequently, the vanadyl organo-mineral compound thus obtained has as aformula: ##STR14##

EXAMPLE 2

The vanadyl sulfate is coupled with the cystein octylamine correspondingto the above mentioned method (iii).

The compound obtained is a complex (L) of vanadyl and of the cysteinoctylamide with the formula: ##STR15##

The above defined vanadyl organo-mineral compound is used in particularin the pharmaceutical industry. More particularly, it has been shownthat a pharmaceutical composition containing as an active substance sucha complex has an anti-diabetic activity and is non-toxic. Thus, suchabove described vanadyl organo-mineral derivatives are therapeuticsubstitutes of the insulin, which can be taken orally in the case ofinsulin-depending diabetes.

First, the potential toxicity of the complex has been investigated.

The investigation has been carried out starting from the bimolecularvanadyl compound having one free amine group, of the general formula(II). After a unique oral administration to the rat, the measuredtoxicity of the compound of formula (II) on the one hand and of thevanadyl sulfate on the other hand have been compared.

The procedure of the toxicological tests is as follows.

On the one hand, the toxicity of the compound (II) is examined byadministering orally increasing doses of compound (II), up to 1 g/kg, toa group of 10 male rats of the WISTAR strain with a mean weight of 150g. A quantity of 10 ml/kg of a suspension of compound (II) in 3% gumarabic is administered, by forcible feeding, to animals which have notbeen fed. Then, the animals are examined for 14 days, in order to countfor the deaths and to search and observe the signs of toxicity.

On the other hand, the acute toxicity of the vanadyl sulfate (VOSO₄) iscontemplated under similar conditions on animals divided into 7 groupsof 10 animals to which the following doses have been administered: 300,400, 500, 600, 800, 1000 and 1500 mg/kg.

The lethal dose (LD₅₀) has been calculated by the method of LITCHFIELDAND WILCOXON.

The results are as follows.

On rats which have absorbed compound (II), no visible sign of toxicityis observed up to the dose of 1 g/kg. The dose of 1 g/kg corresponds toabout 20 times the active anti-diabetic dose.

On rats which have absorbed the vanadyl sulfate, the LD₅₀ is set at 600mg/kg. The first signs of toxicity are already observed at the dose of400 mg/kg. The toxicity appears in the form of a diarrhoea with avariable seriousness, a decrease of the motor activity and a loss offlesh. For the higher doses: 800, 1000 and 1500 mg/kg, hypothermia andcases of chromodacryorrhoea are observed.

Therefore, the vanadyl organo-mineral compound is not toxic whencompared to the vanadyl sulfate, for administration doses of thecompound up to 1 g/kg.

Then, the anti-diabetic activity of the above described complex ofvanadyl and cystein has been examined.

The examination has been performed also starting from the compound withthe formula (II). The activity of this compound has been compared ondiabetic rats and on non-diabetic rats, and the activity of thiscompound (II) has been compared with that of the vanadyl sulfate.

Tests have been carried out on animals having a glycemia higher than 3g/l.

The method is as follows. Diabetes is induced by intraveinous injectionof a unique dose of streptozotocin of about 50 mg/kg, on male or femalerats of the WISTAR strain with a mean weight of 200 g. The glycemia ismeasured 4 days after the injection.

The procedure of the pharmacological tests is as follows. A quantity of45 mg/kg of compound (II) is administered by forcible feeding (doses of10 ml/kg) to animals which have been fed, in the form of a suspension ina 3% solution of gum arabic. A dose of vanadyl sulfate equimolar to 45mg/kg of compound (II) is administered.

The glycemia is measured by quantitative analysis. Blood is sampled fromthe tail of the animal. The glycemia is measured by means of a devicenamed "AMES glucometer" (trade-mark). The rate of glycemia is read on"Derotrostix" dipsticks (trade-mark) of the aforesaid device.

Besides, the insulinemia is measured with a radio-immunological kitnamed "AMERSHAM" (trade-mark).

The results are as follows.

Referring to FIG. 2, there is shown the effect of a uniqueadministration of compound (II) with a dose of 45 mg/kg on the glycemiaand on the insulinemia of diabetic or non-diabetic animals.

On FIG. 2, corresponds to the effect of the oral administration of 45mg/kg of compound (II) on the glycemia of non-diabetic animals;corresponds to the effect of the oral ration of 45 mg/kg of compound(II) on the glycemia of diabetic animals; corresponds to the effect ofthe oral administration of 45 mg/kg of compound (II) on the insulinemiaof non-diabetic animals; corresponds to the effect of the oraladministration of 45 mg/kg of compound (II) on the insulinemia ofdiabetic animals.

On the diabetic animals, the administration of compound of formula (II)induces a drop of the glycemia for about 24 hours. The maximum effect islocated between the fifth and the seventh hour after the administration.After 7 hours, the glycemia decreases progressively and attains a normalrate such as that of non-diabetic animals.

It is to be noted here that the glycemia of non-treated diabetic ornon-diabetic animals does not deviate from the base value of more than0.2 g/l during the experimental period contemplated.

Moreover, it is to be noted also that the administration of a vanadylsulfate dose equimolar to 45 mg/kg of compound (II) induces a weaker andtransitory decrease of the glycemia of diabetic animals. Indeed, themaximal decrease is of about 1 g/l and the duration of the effect islimited to 2 hours after the administration.

Consequently, the vanadyl organo-mineral compound, when administeredorally at a non -toxic dose, presents hypoglycemic properties on thediabetic animals only. The action of this compound is prolonged in thetime and is higher than that of the vanadyl sulfate.

Concerning the insulinemia, FIG. 2 shows that it decreases or remainsstable on diabetic or non-diabetic animals.

Therefore, the administration of compound (II) does not induce theproduction of insulin. There is no hyperinsulinemia. On the contrary, adrop of insulinemia is observed. Under these conditions, the abovedescribed vanadyl organo-mineral compound replaces the insulin whichpresents a surprising advantage. Indeed, the anti-diabetic action of thecompound is not coupled with an increase of the insulinemia.

In view of the foregoing, the complexing of the vanadyl ion by a cysteinderivative permits obtaining a higher anti-diabetic activity and a lowertoxicity with respect to that of vanadyl sulfate, and is not coupledwith a hyperinsulinemia.

Thus, pharmaceutical compositions are prepared from the above describedcomplex as the active substance. Besides the vanadyl organo-mineralderivative, these pharmaceutical compositions comprise excipients.

They are either in a solid form such as capsules, pills or the like, orin a soluble form such as a suspension or drops or the like.

The patients suffering from insulin-depending diabetes can then have anoral treatment and therefore avoid any obligation in relation with aparenteral treatment, without any risk of toxicity.

What is claimed is:
 1. A pharmaceutical composition in a solid form such as capsules, pills comprising as an active substance a vanadyl organo-mineral compound in the form of a complex of a vanadyl and of cysteine with the following general formula: ##STR16## wherein n, p and m are integers equal to 1 or 2 respectively and wherein when p=1 then: Y is selected from the group consisting of a hydrogen atom and a R'-CO group; and(a) when n=1 and m=2, X is selected from the group consisting of an OH group, an OR group and a NHR group wherein R is selected from the group consisting of an alkyl group different from a methyl group comprising from 2 to 9 carbon atoms; wherein: when X=an OH group; Y≦a R'-CO group wherein R' is selected from the group consisting of an alkyl group comprising from 2 to 9 carbon atoms, and, when X is selected from the group consisting of an OR group and an NHR group; Y is H; and (b) when n=2 and m=1, X selected from the group consisting of: ##STR17## wherein R is selected from the group consisting of an alkyl group other than butyl comprising from 2 to 9 carbon atoms; and wherein p=2 then: n=1, m=1, X=an OH group, Y is selected from the group consisting of ##STR18## wherein R' is selected from the group consisting of an alkyl group comprising from 2 to 9 carbon atoms.
 2. A pharmaceutical composition according to claim 1, useful for obtaining an oral anti-diabetic medicine. 